HIV-1 is the causative agent of AIDS. Three viral enzymes reverse transcriptase (RT), integrase (IN), and protease (PR) have essential roles in the replication of HIV-1. We are engaged in a long-term effort to study HIV-1 RT, with the expectation that this information will be useful in the development of more effective anti-RT drugs. Our strategy has involved the analysis of both wild-type and mutant HIV-1 RTs, including drug-resistant mutants. Some of this purified RT has been used by our long-term collaborator, Dr. Eddy Arnold (Rutgers University), for structural studies. We have used purified HIV-1 RT to study the biochemical properties of RT mutants, including drug-resistant mutants. There are two clinically important classes of inhibitors of HIV-1 RT: nucleoside analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). Both are used to treat HIV-1 infections; however, there are serious problems with drug toxicity and with the development of resistance. A major focus of our work on HIV-1 RT is the mechanism(s) of RT inhibitor resistance. We will continue to investigate the mechanisms that underlie drug resistance; however, we have begun to direct a part of our efforts to the development of novel inhibitors that will be effective against the known drug-resistant RTs. We have a collaboration with a skilled nucleoside chemist, Dr. Victor Marquez (Laboratory of Medicinal Chemistry, NCI); the goal of this collaboration is to develop nucleoside analogs that are relatively resistant to excision by NRTI-resistant HIV-1 RTs that are excision proficient. A part of our collaboration with Dr. Arnold is intended to develop more effective NNRTIs. All of the experiments designed to understand NRTI and NNRTI resistance and to develop more effective inhibitors involve a combined structural/biochemical approach; what we have learned from RT structure has been an invaluable guide for planning the biochemical studies, while the results and hypotheses generated in the biochemical experiments have inspired new rounds of structural experiments. Drug resistance is only one aspect of the behavior of RT. We also want to understand how RT carries out reverse transcription in an infected cell, and to correlate the wealth of structural and biochemical data on HIV-1 RT with the actual process of reverse transcription. These experiments are part of Project Z01 BC 010482 (Retroviral Replication and Vector Design). In some cases, it will not be possible to explain the in vivo data with the available structural and biochemical results. In such cases, we will do additional biochemical and structural experiments to complement (and better understand) the in vivo results. [Corresponds to Hughes Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]